Biomedical subjects
Publications and source records attributed to S V Fuggle.
During a study of MICA frequency in a healthy population and a cohort of patients suffering with inflammatory bowel disease, three DNA samples produced unusual reactivity patterns using polymerase chain reaction sequence-specific primers (PCR-SSP). These samples were subsequently characterized by sequence-based typing (SBT). Here, we report the sequence of these three novel MICA alleles.
We have identified a novel MICA allele, MICA*051, detected by the polymerase chain reaction using sequence-specific primers and characterized by sequence-based typing. MICA*051 appears to be the result of a recombination between MICA*00801 and MICA*00701 at intron 2.
We report the identification of a novel major histocompatibility complex class I-related chain (MICB) allele, provisionally designated as MICB-0114 pending the WHO Nomenclature Classification for the MICB locus. This new allele is identical to MICB-0103101v except for a single mutation of G to A in exon 4 that translates into an amino acid substitution from glutamic acid to lysine.
Alloimmunization to human platelet alloantigens (HPAs) is responsible for neonatal alloimmune thrombocytopenia (NAIT), post-transfusional purpura (PTP) and platelet transfusion refractoriness. HPAs may also have a role as histocompatibility antigens in transplantation as well as associations with cardiac disease. We have developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) assay capable of detecting 15 HPA allelic variants. As part of the validation of the assay, 134 UK renal donors were genotyped to determine HPA allele frequencies in the UK population. The HPA allele frequencies obtained are consistent with those of the other European studies: GP1A*1 (HPA-5a) and GP1A*2 (HPA-5b), 0.914 and 0.086, respectively; GP1BA*1 (HPA-2a) and GP1BA*2 (HPA-2b), 0.925 and 0.075; GP2B*1 (HPA-3a) and GP2B*2 (HPA-3b), 0.627 and 0.373; GP3A*1 (HPA-1a) and GP3A*2 (HPA-1b), 0.840 and 0.161. The rare alleles GP2B*3 (HPA-9bw) and GP3A*3 to *8 (HPA-4b, -6b, -7bw, -8bw, -10bw and -11bw, respectively) were all absent. This comprehensive HPA genotyping assay allows rapid, accurate and reproducible results at low cost.
The presence of an unusual HLA class I reactivity pattern was detected in a Caucasoid-Asian individual by PCR-sequence specific primer (PCR-SSP) typing. Exons 2 and 3 were characterized using PCR-sequence-based typing (PCR-SBT) and were found to contain a novel Cw*03 sequence, Cw*0315. In the region studied, Cw*0315 was comprised mainly of the Cw*0302 sequence, but at four positions it contained nucleotides normally only found in other HLA Cw locus alleles. These positions each resulted in an amino acid substitution.
Hereditary hemochromatosis (HH) is an iron-overload disease common in populations of Northern European origin. Patients display increased iron absorption leading to excessive iron deposition and potential multiorgan failure. Using polymerase chain reaction sequence-specific primer (PCR-SSP) technology, we have developed an HH diagnosis assay capable of detecting 19 non-synonymous HFE mutations (including a previously unreported mutation, V295A) and several TFR2, SLC11A3 and H ferritin alleles implicated in HH. As part of the validation process, 159 UK renal donors were genotyped to determine HH allele frequencies in the UK population. The alleles nominally identified as HFE*01 (C282Y), HFE*02 (H63D) and HFE*03 (S65C) were found at frequencies of 0.085, 0.173 and 0.009, respectively. All other potential HH-associated alleles were absent, confirming their low prevalence in this population. This assay enables comprehensive routine HH genotyping, producing rapid, accurate and reproducible results at low cost.
BACKGROUND: Organs used for transplantation may experience long periods of cold ischemic preservation and consequently oxygen free radical-mediated damage following reperfusion. Lecithinized superoxide dismutase (lec-SOD) is a novel free radical scavenger that has been shown to bind with high affinity to cell membranes. The aim of this study was to determine whether lec-SOD bound to endothelial cells under organ preservation conditions to mediate direct antioxidant activity at the endothelial cell surface and thus offer protection against the harmful effects of ischemia/reperfusion injury. METHODS: An in vitro study was performed on large vessel endothelial cells (HUVEC) and a human microvascular endothelial cell line HMEC-1, to investigate the potential therapeutic benefits of incorporating lec-SOD into organ preservation solution. A cold hypoxia/reoxygenation system was developed to examine lec-SOD binding affinity to endothelial cells, protection against hypoxia/reoxygenation-induced cell death, and neutrophil adhesion. RESULTS: Lec-SOD bound to endothelial cells with higher affinity than unmodified recombinant human superoxide dismutase (rhSOD) and significantly protected both HUVEC and HMEC-1 from cell death following 27 hours of cold hypoxia (P < 0.01). Furthermore, neutrophil adhesion to the endothelium stimulated by hypoxia and reoxygenation was significantly inhibited by treatment with lec-SOD but not by lecithin or rhSOD (P < 0.01). Analysis by flow cytometry demonstrated that E-selectin and ICAM-1 were up-regulated by hypoxia/reoxygenation that was inhibited in part by lec-SOD. CONCLUSIONS: The results from this study suggest that incorporation of lec-SOD into organ preservation solutions provides effective protection to endothelial cells against cold ischemia and reperfusion injury following transplantation.
We have identified an HLA-B*07 variant allele, B*0716, in a Caucasoid cadaver kidney donor. The HLA class I type by polymerase chain reaction using sequence-specific primers (PCR-SSP) was A*01, 32; B*07, 08; Cw*07. Serological typing, using monoclonal and polyclonal anti-HLA antisera, gave disparate results for the B antigens. Monoclonal antibodies identified B7 and B8 antigens but polyclonal antisera recognised only the B8 antigen. PCR using sequencing based typing (PCR-SBT) confirmed the presence of both B*0703 and B*0801 alleles but with a mutation in one of the alleles. The HLA-B*07 allele was isolated by allele-specific PCR and was shown to have a mutation, G-->T, at 292 in exon 2. This mutation changes codon 74, which encodes aspartic acid (GAC) present in all previously identified B*07 alleles, to tyrosine (TAC) in the variant. The serological results suggest that codon 74 is a crucial part of a B7 antigen-specific epitope recognised by tissue typing polyclonal antisera.
OBJECTIVE: To identify potential risk factors for the development of chronic renal allograft failure. SUMMARY BACKGROUND DATA: Chronic allograft failure (CAF) is the leading cause of late graft loss in renal transplantation. The authors studied the risk factors for the development of CAF in a single center during a period in which a consistent baseline immunosuppression regimen (cyclosporine, azathioprine, and prednisolone) was used. METHODS: Data from the Oxford Transplant Center Database were assessed on 862 renal allografts during a 10-year period. Risk factors were identified using multivariate logistic regression analysis. RESULTS: Biopsy-proven CAF occurred in 77 patients (9.2%) in the entire group. Multivariate risk factor analysis revealed that early and late acute rejection episodes, proteinuria, and serum triglycerides were significant factors. Acute rejection after 3 months was more important than early acute rejection. Serum triglyceride level and proteinuria at 1 year were both elevated in the CAF group. Male sex provided a protective effect. Serum creatinine levels at 6 months after the transplant were not predictive of the risk of developing CAF. CONCLUSIONS: These results from the largest single-center review to date suggest that both antigen-dependent and -independent factors are involved in the pathogenesis of CAF. Acute rejection at all time points has a significant impact on the development of CAF.
RENAL TRANSPLANT OUTCOME: Analysis of 5-year transplant survival in the UK showed a number of significant factors influencing outcome of adult cadaveric renal transplantation. Data from 5,963 first grafts and 1,078 regrafts carried out between 1990-1997 showed year of graft, recipient age and diabetes, donor age, kidney exchange between centres and HLA matching to influence 5-year outcome. The most important prognostic factor was donor age: the risk of transplant failure within 5 years for grafts using kidneys from donors aged 60 years and over was double that of grafts using donors aged 18-34 years. Unlike the effect of donor age, the influence of HLA matching would appear to be diminishing with time. In contrast to transplants in the 1980's, the difference in 5-year transplant survival between 000 mismatched and favourably matched (100, 010 or 110 mismatched) transplants is no longer significant. An analysis of posttransplant survival for first grafts in different epochs (0-3 months, 3 months to 3 years and beyond 3 years) showed that one factor affected short-term outcome (exchange of kidneys between centres), whereas others affected outcome throughout the epochs (most notably donor age, recipient age and recipient diabetes). RECIPIENT AND DONOR AGE MATCHING: The mean recipient age in the UK and Republic of Ireland increased by 5 years between 1981-1990 but has remained at approximately 45 years since then. The mean donor age increased by 7 years to 42.5 years (s.e. 0.5) between 1981-1991 and since then has increased at a slower rate to 43.4 years (s.e. 0.5) in 1998. The mean donor-recipient age difference for more than 15,000 transplants carried out between 1990-1998 has decreased, primarily due to increasing donor age over this time. However, the introduction of a new Kidney Allocation Scheme in the UK in July 1998, part of which is aimed at minimising age differences, has increased the likelihood that recipients aged over 60 years will be allocated grafts from donors closer to their own age than previously. The new UK Kidney Allocation Scheme also gave children increased access to well-matched adult organs leading to an increased mean age difference for this group between July-December 1998. DONOR AND RECIPIENT HLA MATCHING: Modifications to the Kidney Allocation Scheme introduced in January 1997 with the aim of increasing the number of well-matched transplants has led to a rise in 000 mismatched grafts from 5% to 7% and favourably matched (100/010/110 mismatches) from 29% to 36% between 1990-1992 and 1996-1998. Over this same time the proportion of 2 DR-mismatched grafts has decreased from 10% to 4%. The revised Kidney Allocation Scheme implemented in July 1998 gave a further increase in priority to 000 mismatches, increasing the proportion of these transplants to 12% for the last half of 1998, a level which has been maintained since then.
BACKGROUND: In the UK, kidneys are exchanged between centres on the basis of matching for HLA. We analysed various factors that might affect graft outcome to establish whether exchange of kidneys on this basis remains valid. METHODS: 6363 primary cadaveric renal transplants carried out in 23 centres in the UK between 1986 and 1993 were used in the analysis. 6338 (99.6%) patients who underwent transplantation were followed up at 1 year. 5-year follow-up data were available for 2907 (97.8%) of the 2972 patients who survived to 5 years. We made random checks to validate the data. A multifactorial analysis with Cox's proportional hazards models was used to analyse factors that had a possible effect on graft outcome. To ensure that the analysis of matching was constant during the 8-year study, our analysis was based on the HLA antigens used for organ exchange (11 A locus antigens, 27 B locus antigens, and 12 DR locus antigens). We assessed overall outcome at 5 years and during three periods after transplantation at: 0-3 months, 3-36 months, and after 36 months. FINDINGS: The following factors were significantly associated with graft outcome in the multifactorial analysis: year of graft, age of donor, age of recipient, whether the recipient had diabetes, cause of donor's death, cold ischaemic time, transport of kidneys, transplant centre, and matching for HLA. The best outcome was achieved with kidneys that had no mismatches at HLA-A, HLA-B, and HLA-DR loci (000 mismatches). The next most favourable outcome was achieved with one mismatch at either A or B loci or one mismatch at both the A and B , but no mismatch at the DR locus (100, 010, or 110 mismatches). Age of the donor and recipient had a significant effect on transplant outcome: older age was associated with increased risk of graft failure. INTERPRETATION: Various factors affect the outcome of primary cadaveric renal transplantation, particularly the age of the donor and the recipient. However, the effect of matching for HLA remains a strong one and fully justifies the continuing policy in the UK of exchanging kidneys on the basis of HLA matching, especially to recipients when there is a 000 mismatch for HLA between donor and recipient. On the basis of this analysis, a new allocation scheme for kidneys was introduced in the UK in 1998. During the first 9 months of the scheme, there has been a doubling of the number of HLA-000 mismatched kidneys transplanted.
It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.
Delayed graft function (DGF) and acute rejection have both been associated with reduced renal allograft survival. In some studies, they have been shown to have an interactive effect. We studied the risk factors for DGF and the relative impact of DGF and rejection on both short- and long-term survival in recipients of cadaveric renal transplants. Data from the Oxford Transplant Centre Database were assessed on 710 cadaver allografts over a 10-yr period, during which time all recipients received cyclosporin-based immunosuppressive protocols. The interaction between DGF and acute rejection was examined using logistic and Cox multivariate regression. Long cold ischaemia time (CIT), sensitisation and older donor age were found to be independent predictors of DGF. The occurrence of DGF resulted in a reduced 5-yr survival (56 vs. 75%). However, the effect of DGF was confined to the first year post-transplant, as there was no significant difference in survival, as measured by half-life (t1/2) of grafts functioning at 1 yr, with DGF alone and a group with good early function (t1/2 = 21.3 vs. 20.0 yr). There was no increase in acute rejection in grafts with DGF. However, the combination of DGF and acute rejection resulted in the worst short-term graft survival (68% at 1 yr, compared to 92.3% in those grafts with no DGF or acute rejection) and this continued over the long term (t1/2 = 10.5 yr). These data suggest that early function is critical to the success of renal transplantation. The effects of DGF are limited to the first year post-transplant. Long-term graft survival may be improved by efforts to limit CITs, particularly for grafts from older donors and sensitised recipients.
We have identified an HLA-A11 variant allele, A*1105, segregating in a Caucasoid family. The variant antigen expressed by this allele failed to cross-react with most Caucasoid anti-HLA-A11 antisera tested. Sequencing based typing has been used to characterize this new allele and this showed that it has a novel mutation at a polymorphic position (502) in exon 3. In comparison with A*1101, the mutation (A-->G) results in an amino acid change from positively-charged lysine to negative glutamate and this may explain the altered HLA-A11 serological profile exhibited by this antigen. The new allele was found in a patient with acute lymphoid leukaemia (ALL), her father and two siblings.
The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross-species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non-leukocyte cross-reactivity. A total of 63 out of 127 antibodies cross-reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross-reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM1O1A, K20) and CD18 (BU87, 7E4), the common beta1- and beta2-integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross-species reactivity of human antibodies, it is important to consider the possibility that there may be additional non-leukocyte reactivity in other tissues.
BACKGROUND: Chronic allograft failure (CAF) is a major cause of late graft loss in renal transplantation. Up-regulation of adhesion molecules has been demonstrated in renal allograft biopsies during both acute and chronic rejection, and these molecules are known to regulate leukocyte migration into the graft. METHODS: A single-center retrospective study was performed between 1985 and 1996 on renal transplant recipients who developed CAF. Genotyping was performed for five polymorphisms in intercellular adhesion molecule-1 (ICAM-1), E-selectin, and L-selectin. Frequency data for the polymorphisms in the CAF group (N = 62) and their matched donors, where available (N = 33), were compared with a group of recipients with graft survival of more than 10 years (N = 110) and a group of United Kingdom (UK) controls (N = 101). RESULTS: A variant allele in exon 4 of ICAM-1 (R241) was more common in the CAF recipients compared with both long-term survivors and UK controls (19.4 vs. 10.0 and 9.4%, P = 0.015 and 0.025). In addition, stratification by time to graft failure caused by CAF revealed more rapid failure in the presence of another ICAM-1 variant in the recipient (E469) in exon 6 (P = 0.033). CONCLUSIONS: ICAM-1 polymorphisms may represent a predetermined genetic risk factor for CAF. The polymorphism in exon 4 is in the Mac-1 binding site, and that in exon 6 is in the fifth immunoglobulin-like domain. Potential mechanisms of action of ICAM-1 variants in CAF include an alteration of activity as an adhesion molecule, altered costimulation, or a minor histocompatibility antigen.
BACKGROUND: It is widely recognized that living-related donor (LRD) renal allografts have a higher overall graft survival than cadaver donor transplants. We tested the hypothesis that part of this is attributable to LRD kidneys being obtained under optimal conditions from healthy donors, whereas cadaveric kidneys may have experienced injury as a result of inflammatory events around the time of brain death. METHODS: We have performed a comparative immunohistochemical analysis of pretransplant donor biopsies from cadaveric (N = 65) and LRD (N = 29) kidneys to determine any differences that may predispose them to subsequent damage. Cryostat sections were stained with antibodies to leukocytes, adhesion molecules, and human leukocyte antigen (HLA)-DR antigens, and the expression was assessed semiquantitatively. RESULTS: High levels of endothelial E-selectin and proximal tubular expression of HLA-DR antigens, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 were detected in biopsies from cadaveric kidneys, whereas expression of these markers was markedly reduced in LRD kidneys. High levels of tubular antigen expression were significantly associated with traumatic death, prolonged ventilation, and episodes of infection in cadaver donors. Furthermore, the expression of pretransplant tubular antigens in cadaver donor kidneys was significantly associated with early acute rejection following transplantation, suggesting that such kidneys are predisposed to subsequent immune-mediated attack following transplantation. CONCLUSIONS: These results may explain, in part, the superior outcome of LRD allografts compared with cadaver renal allografts.
BACKGROUND: The Banff classification for assessment of renal allograft biopsies was introduced as a standardized international classification of renal allograft pathology and acute rejection. Subsequent debate and evaluation studies have attempted to develop and refine the classification. A recent alternative classification, known as the National Institutes of Health Collaborative Clinical Trials in Transplantation (NIH-CCTT) classification, proposed three distinct types of acute rejection. The 1997 Fourth Banff meeting appeared to move towards a consensus for describing transplant biopsies, which incorporated both approaches. Patients who received a renal allograft at the Oxford Transplant Centre were managed by a combination of protocol and clinically indicated biopsies. We have undertaken a retrospective analysis of the biopsies correlated with the clinical outcome to test the prognostic value of the original Banff (Banff 93-95) and NIH-CCTT classifications. METHODS: Three hundred and eighty-two patients received renal allografts between May 1985 and December 1989, and were immunosuppressed using a standard protocol of cyclosporine, azathioprine and steroid. Adequate 5-year follow-up data were available on 351 patients, and of these, 293 had at least one satisfactory biopsy taken between days 2 and 35 after transplantation, the latter patients forming the study group. The D2-35 biopsies taken from these patients, which were not originally reported according to the Banff classification, were re-examined and classified according to the Banff 93-95 protocols. For each patient the biopsy found to be the most severely abnormal was selected, and the Banff and NIH-CCTT grading compared with the clinical outcome. RESULTS: Seven hundred and forty-three biopsies taken from 293 patients between days 2 and 35 after transplantation were examined and the patients categorized on the basis of the 'worst' Banff grading as follows. Normal or non-rejection, 20%; borderline, 34%; acute rejection grade I (AR I), 18%; AR IIA, 6%; AR IIB, 14%; AR III, 1%; AR IIIC, 3%; widespread necrosis 3%. The clinical outcome for the last two groups combined was very poor with 18% of grafts functioning at 3 months and 6% at 5 years. The other groups with vascular rejection (AR IIB and AR III) had an intermediate outcome, graft survival being 78% at 3 months and 61% at 5 years. The remaining four groups (normal, borderline, cellular AR I and AR IIA) had the best outcome: graft survival 95% at 3 months and 78% at 5 years with virtually no difference between the four groups. Three forms of acute rejection, namely tubulo-interstitial, vascular and transmural vascular, were identified, but only the latter two categories were associated with a poor outcome. CONCLUSIONS: The eight sub-categories of the Banff classification of renal allograft biopsies are associated with three different prognoses with respect to graft survival in the medium term. These three prognostic groups correspond to the three NIH-CCTT types. The data provide support for the consensus developed at Banff 97 separating tubulo-interstitial, vascular and transmural vascular rejection (types I, II and III acute rejection).